This invention relates to the isolation and cloning of neutral protease genes and the expression of the cloned genes in gram negative bacteria. More specifically, genes coding for neutral proteases have been cloned and expressed, with confirmation that the clones synthesize and secrete active neutral protease enzyme.
Vibrio proteolyticus (Aeromonas proteolytica), which contains in its genome one or more genes for neutral protease, has been identified and cultured. Griffin et al., "Some Physical Characteristics of a Proteinase from Aeromonas proteolytica," J. Biol. Chem., Vol. 245, pp. 1348-56 (1970), describes a proteinase isolated from culture filtrates of A. proteolytica. Bayliss et al., Archives of Biochemistry and Biophysics, 204:214-219, (1980), isolated and identified a neutral protease from Aeromonas proteolytica. Neutral proteases also have been prepared by cultivating various Bacillus strains, as taught in U.S. Pat. No. 3,796,635 (Delente).
Purification of enzymes for industrial uses is hampered by the typically low levels of enzyme produced by naturally occurring isolates. Using genetic engineering to manipulate the gene coding for an enzyme of interest, the gene can be relocated into other organisms for both laboratory development and industrial production of the enzyme. Drawbacks associated with production of the enzyme in its natural environment can thereby be avoided.
Previous attempts at cloning related protease enzymes in E. coli have yielded varying results. For example, Yanagida et al., "Specific Excretion of Serratia marcescens Protease through the Outer Membrane of Escherichia coli," J. Bacteriology, 166:937-44 (1986), discloses cloning of a serine protease DNA fragment from the microorganism Serratia marcescens into E. coli in which there was specific secretion of the protease into the extracellular medium. By contrast, Nakahama et al., "Cloning and Sequencing of Serratia Protease Gene," Nucleic Acids Research, 14:5843-55 (1986), found no excretion upon cloning the Serratia sp. E-15 extracellular metalloproteinase gene into E. coli, but reported excretion of the protease into the culture medium when the gene was cloned back into Serratia.